We have focused our efforts to elucidate: i) the catalytic cycle and transport pathway of human P-gp; ii) the molecular basis of the polyspecificity of P-gp; iii) the interaction of clinically relevant tyrosine kinase inhibitors (TKIs) and other natural product modulators with P-gp and ABCG2; iv) pharmacophore features required for binding of third generation cyclic peptide inhibitor derivatives (BTT compounds) to P-gp and ABCG2; and vi) the use of single particle cryo-EM for the analysis of the conformational landscape of human P-gp during its catalytic cycle. We have been employing cell-based, biochemical, biophysical, pharmacological, and physiological techniques along with molecular biology and molecular modeling approaches to extend our understanding of the mechanistic aspects and the structure-function relationships of ABC drug transporters. In addition, we have devoted considerable effort to the screening and development of TKIs and small molecule modulators of P-gp and ABCG2 that are used in the clinic for treatment of various types of cancers. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of P-gp and the role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of P-gp. We are using molecular modeling and mutagenesis approaches to elucidate on a molecular level how this transporter recognizes and transports a wide variety of structurally dissimilar compounds. We recently identified a phenylalanine-tyrosine structural motif in the drug-binding pocket of P-gp that is critical for the inhibition of ATP hydrolysis by zosuquidar and other high-affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, we made a global alteration of the drug-binding pocket using gene synthesis to introduce fifteen tyrosine residues in the drug-binding pocket to study its effect on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine, two leucine, one isoleucine, and one methionine. Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size fluorescent substrates, its ability to transport large ( above 1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased, thus revealing a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. To our knowledge, this is the first report demonstrating that the global alteration of the drug-binding pocket with substitution of fifteen residues is well tolerated. The 15Y mutant has six mutated residues located in TMD1 and nine in TMD2. The generation of mutants with either six substitutions in TMD1 or nine in TMD2 will be useful to assess the role of each TMD of the transporter and to narrow the number of residues contributing to substrate size determination. 2. The mechanism of the molecular basis of polyspecificity, which is an important property of multidrug transporters, by using molecular modeling and mutagenesis approaches: Towards the goal of understanding the molecular basis of broad substrate specificity of P-gp, we characterized the effect of a detergent micelle environment on the drug-mediated inhibition of P-gp ATPase activity. Most of the substrates or modulators of P-gp stimulate its basal ATPase activity, and only a few drugs have been found to inhibit it. Several structural studies on purified P-gp have been reported and there is very limited and in some cases conflicting information available on ligand interactions with isolated transporters in a dodecyl maltoside detergent environment. For these reasons, we compared the biochemical properties of human and mouse P-gp in native membranes, detergent micelles, and after reconstitution in artificial membranes. We found that the modulators zosuquidar, tariquidar and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment, whereas these drugs inhibited the ATPase activity of the transporter in native membranes or when it was reconstituted in proteoliposomes. Similarly, a 30- to 150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared to native or artificial membranes. These findings in aggregate demonstrated that the high-affinity site is inaccessible either due to a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. Thus, the structural and functional aspects of ligand interactions with purified P-gp need to be studied in a detergent-free native or artificial membrane environment. 3. Resolution of the three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of P-gp is an ongoing project and for this we have developed a purification scheme that has yielded total protein of 7.5-10.0 mg of 99% homogeneously pure P-gp. Due to the flexible nature of human P-gp, we are also using single particle analysis by the cryo-electron microscopy technique. To improve the resolution to an atomic level, we have begun to use the purified P-gp reconstituted in nanodiscs. We have optimized the conditions for reconstituting purified P-gp in nanodiscs in a functional form. The ratio of membrane scaffold protein (MSP) 1 to purified P-gp and the lipid composition of nanodiscs is critical to retain the function of purified protein in nanodiscs. The properties of P-gp in nanodiscs are similar to those observed in native membranes. The EM analysis of negatively stained P-gp-nanodisc samples in the absence and presence of ATP-Mg, ADP-vanadate and Fab of UIC2 antibody suggests that there is a single molecule of P-gp per nanodiscs. The single particle cryo-EM studies are carried out in collaboration with Dr. Sriram Subramaniam (LCB). 4. Development of non-toxic natural product and small molecule modulators to overcome resistance mediated by P-gp and ABCG2: We have extended these studies by synthesizing over 100 derivatives of the cyclic peptide inhibitor TTT28 in collaboration with Dr. Tanaji Talele (St. John's University, NY). We found that at least two derivatives inhibited the ATPase activity of P-gp and sensitized cells expressing P-gp and ABCB2 to anticancer drugs. The docking studies indicated that these derivatives bind to the drug-binding pocket of P-gp. To validate the docking poses of one of the inhibitory derivatives (compound 109), we used single or double mutations in the drug-binding pocket. In addition, we continue to characterize the recently developed tyrosine kinase inhibitors, which are used in the clinic to treat cancer patients, for their effect on the function of P-gp and ABCG2. The phosphatidylinositol 3-kinase inhibitor PF-498216 was found to modulate the function of ABCG2, whereas regorafenib was found to modulate only the function of P-gp. We also found that the BTK inhibitor, ibrutinib (PCI-32765) can overcome the resistance to paclitaxel-mediated by P-gp and ABCC10 (MRP7) in a mouse xenograft model. These studies were carried out in collaboration with Drs. Chug-Pu Wu (Chang Gung University, Taiwan) and Zhe-Sheng Chen (St. John's University, NY).